Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of each series of experiments.
The classical method for developing monoclonal antibodies specific for cell-surface molecules involves repeated injections of mice with either intact cells or cell membrane preparations derived from the desired target cells. The injections are followed by the removal of mouse spleen cells and fusion of these cells to a myeloma partner [reviewed in (1-3)]. This approach has resulted in the production of monoclonal antibodies that react with a number of surface-expressed molecules of potential interest, including cell-surface growth factor receptors and tumor-associated antigens. However, the procedure is generally inefficient and requires screening of a large number of hybridomas for production of the appropriate monoclonal antibodies [reviewed in (1-6)].
DNA transfection procedures have been used to transfer human genes into heterologous cells, such as mouse NIH 3T3 cells [reviewed in 7-10)]. When NIH 3T3 cells have been used as the recipient for DNA transfection, this approach has not been successful in identifying dominant-acting transforming or tumor-inducing genes from a majority (approximately 85%) of human tumors or human tumor cell lines (7-10). In most studies that use NIH 3T3 cells, even when a dominant-acting oncogene was identified, it often represented a member of the ras oncogene family or a modified cellular gene (7-10). A recently developed cloned rat embryo fibroblast cell line, CREF-Trans 6, has proven useful in identifying putative novel oncogenes not detected in NIH 3T3 cells (11). Cotransfection of CREF-Trans 6 cells with high-molecular-weight DNA from the LNCaP human prostatic carcinoma cell line and the selectable neomycin resistance gene (pSV2neo), followed by selection for resistance to G418 and injection into nude mice, resulted in tumor formation (11). In contrast, when the same DNA sources were used with NIH 3T3 cells, no tumors developed in nude mice given an injection of neomycin-resistant (G418) cotransfected NIH 3T3 cells (11).
Applicants conducted the current experiments to determine if DNA transfection combined with an immunologic masking tactic could be used to efficiently generate hybridomas that secrete monoclonal antibodies reacting with cell-surf ace molecules expressed on genetically altered cells. Applicants demonstrate the feasibility of this approach, called surface-epitope masking (SEM). Applicants used DNA transfection and the SEM procedure in an attempt to produce hybridomas secreting monoclonal antibodies that reacted with surface epitopes located on typical multidrug-resistant (MDR) cells and human prostatic carcinoma cells. These results indicate that DNA transfection in conjunction with SEM can be used to generate hybridomas producing monoclonal antibodies that can react with surface-expressed molecules encoded by both known and unknown genes.
This invention provides a method for preparing a hybridoma cell line which produces an antibody capable of specifically binding to a cell surface-expressed protein which expresses on the surface of one cell type but not the other comprises a) generating antiserum against a cell type which does not express the cell surface-expressed protein; b) coating another cell type which expresses the cell surface-expressed protein with the antiserum generated; c) injecting the antiserum-coated cells into suitable hosts; d) screening the resulting hosts to identify hosts which produce serum reactive with the coated cell; e) removing spleens from the hosts so identified; f) preparing from the spleens so removed hybridomas; and g) recovering therefrom a hybridoma cell line which produces an antibody capable of specifically binding to a cell surface-expressed protein.
This invention provides a method for isolating DNA coding for a protein capable of binding to the cell surface-expressed protein which expresses on the surface of one cell type but not the other comprising: a) generating antiserum against a cell type which does not express the cell surface-expressed protein; b) coating another cell type which expresses the cell surface-expressed protein with the antiserum generated; c) injecting the antiserum-coated cells into suitable hosts; d) screening the resulting hosts to identify hosts which produce serum reactive with the coated cell; e) removing spleens from the hosts so identified; f) isolating B-lymphocytes from the removed spleen; g) preparing DNA from plasma cells to generate combinatorial phage cDNA library which contains different clones; and h) contacting the clones in the library with the coated cells from step (b), the binding of the coated cells with a clone indicating the protein expressed by the clone capable of binding to the cell surface-expressed protein.
This invention provides a method for preparing a hybridoma cell line which produces an antibody capable of specifically binding to a cell surface-expressed protein which expresses on the surface of one cell type but not the other comprises: a) generating antiserum against a cell type which does not express the cell surface-expressed protein; b) coating another cell type which expresses the cell surface-expressed protein with the antiserum generated; c) contacting the antiserum-coated cells with suitable immunoresponsive cells capable of being stimulated to produce antibodies; d) preparing immunoresponsive cells to produce hybridomas; and e) isolating hybridomas which produce antibodies reactive with the coated cell, thereby preparing hybridoma cell lines which produce antibodies capable of specifically binding to a cell surface-expressed protein.
This invention provides a method for preparing a hybridoma cell line which produces an antibody capable of specifically binding to a cell surface-expressed protein comprises: a) generating antiserum against a cell which normally does not express the cell surface-expressed protein; b) introducing a DNA molecule encoding the cell surface-expressed protein to express the cell surface-expressed protein into the cell; c) selecting cells which express the cell surface-expressed protein; d) coating the selected cells with the antiserum generated in step a; e) injecting the antiserum-coated cells into suitable hosts; f) screening the resulting hosts to identify hosts which produce serum reactive with the coated cell; g) removing spleens from the hosts so identified; h) preparing from the spleens so removed hybridomas; and i) recovering therefrom a hybridoma cell line which produces an antibody capable of specifically binding to a cell surface-expressed protein.
This invention provides a method for isolating DNA coding for a protein capable of binding to the cell surface-expressed protein comprising: a) generating antiserum against a cell which normally does not express the cell surface-expressed protein; b) introducing a DNA molecule which encodes the cell surface-expressed protein to express the cell surface-expressed protein into the cell; c) selecting cells which express the cell surface-expressed protein; d) coating the selected cells with the antiserum generated in step a; e) injecting the antiserum-coated cells into suitable hosts; f) screening the resulting hosts to identify hosts which produce serum reactive with the coated cell; g) removing spleens from the hosts so identified; h) isolating B-lymphocytes from the removed spleen; i) preparing DNA from B-lymphocytes to generate combinatorial phage cDNA library which contains different clones; and j) contacting the clones in the library with the coated cells from step (b), the binding of the coated cells with a clone indicating the protein expressed by the clone capable of binding to the cell surface-expressed protein.
This invention provides a method for, preparing a hybridoma cell line which produces an antibody capable of specifically binding to a cell surface-expressed protein which expresses on the surface of one cell type but not the other comprises: a) generating antiserum against a cell which normally does not express the cell surface-expressed protein; b) introducing a DNA molecule which encodes the cell surface-expressed protein to express the cell surface-expressed protein into the cell; c) selecting cells which express the cell surface-expressed protein; d) coating the selected cells with the antiserum generated in step a; e) contacting the antiserum-coated cells with suitable immunoresponsive cells capable of being stimulated to produce antibodies; f) preparing immunoresponsive cells to produce hybridomas; and g). isolating hybridomas which produce antibodies reactive with the coated cell, thereby preparing hybridoma cell lines which produce antibodies capable of specifically binding to a cell surface-expressed protein.
This invention provides a method for preparing a hybridoma cell line which produces an antibody capable of specifically binding to a cell surface-expressed protein comprises: a) introducing a DNA molecule which encodes a cell surface-expressed protein and a second DNA molecule which encodes a selectable or identifiable trait into an established cell line; b) selecting transfected cells which express the selectable or identifiable trait; c) recovering the transfected cells so selected; d) coating the selected cells so recovered with an antiserum generated against the established cell line; e) injecting the antiserum-coated cells into the suitable hosts; f) screening the resulting hosts to identify hosts which produce serum reactive with the coated cell; g) removing spleens from the hosts so identified; h) preparing from the spleens so removed hybridomas; and i) recovering therefrom a hybridoma cell line which produces an antibody capable of specifically binding to a cell surface-expressed protein.
This invention provides a method for isolating DNA coding for a protein capable of binding to the cell surface-expressed protein comprising: a) introducing a DNA molecule which encodes a cell surface-expressed protein and a second DNA molecule which encodes a selectable or identifiable trait into an established cell line; b) selecting transfected cells which express the selectable or identifiable trait; c) recovering the transfected cells so selected; d) coating the selected cells so recovered with an antiserum generated against the established cell line; e) injecting the antiserum-coated cells into the suitable hosts; f) screening the resulting hosts to identify hosts which produce serum reactive with the coated cell; g) removing spleens from the hosts so identified; h) isolating B-lymphocytes from the removed spleen; i) preparing DNA from B-lymphocytes to generate combinatorial phage cDNA library which contains different clones; and j) contacting the clones in the library with the coated cells from step (b), the binding of the coated cells with a clone indicating the protein expressed by the clone capable of binding to the cell surface-expressed protein.
This invention provides a method for preparing a hybridoma cell line which produces an antibody capable of specifically binding to a cell surface-expressed protein comprises a) introducing a DNA molecule which encodes a cell surface-expressed protein and a second DNA molecule which encodes a selectable or identifiable trait into an established cell line; b) selecting transfected cells which express the selectable or identifiable trait; c) recovering the transfected cells so selected; d) coating the selected cells so recovered with an antiserum generated against the established cell line; e) contacting the antiserum-coated cells with suitable immunoresponsive cells capable of being stimulated to produce antibodies; f) preparing immunoresponsive cells to produce hybridomas; and g) isolating hybridomas which produce antibodies reactive with the coated cell of step (d), thereby preparing hybridoma cell lines which produce antibodies capable of specifically binding to a cell surface-expressed protein.
This invention provides a method for preparing a hybridoma cell line which produces an antibody which specifically recognizes and binds to a tumor associated antigen associated with a neoplastic, human cell which comprises: a) cotransfecting the CREF-Trans 6 cell line (ATCC Accession No. CRL 10584) with DNA isolated from a neoplastic, human cell and DNA which encodes a selectable or identifiable trait; b) selecting transfected cells which express the selectable or identifiable trait; c) recovering the transfected cells so selected; d)injecting the transfected cells so recovered into a suitable first murine host; e) maintaining the resulting first murine host for a period of time effective to induce the injected transfected cells to form a tumor in the first murine host; f) isolating the resulting tumor from the first murine host; g) obtaining tumor cells from the tumor so isolated; h) coating the tumor cells so obtained with an antiserum generated against the established non-human, non-tumorigenic cell line; i) injecting the antiserum-coated cells into the suitable second hosts; j) screening the resulting second hosts to identify hosts which produce serum reactive with the neoplastic, human cell; k) removing spleens from the second hosts so identified; l) preparing from the spleens so removed hybridomas; and m) recovering therefrom a hybridoma cell line which produces an antibody which specifically recognizes and binds to the cell surface antigen.
This invention provides a method of preparing DNA encoding a cell surface antigen associated with a neoplastic, human cell which comprises: a) cotransfecting CREF-Trans 6 cell line with DNA isolated from a neoplastic human cell and DNA encoding a selectable or identifiable trait; b) selecting transfected cells which express the selectable or identifiable trait; c) recovering the transfected cells so selected; d) injecting the transfected cells so recovered into a suitable first murine host; e) maintaining the resulting first murine host for a period of time effective to induce the injected transfected cells to form a tumor in the first murine host; f) isolating the resulting tumor from the first murine host; g) obtaining tumor cells from the tumor so isolated; and h) recovering DNA encoding the cell surface antigen associated with the neoplastic human cell from the tumor cells so obtained.
This invention further provides an isolated mammalian nucleic acid molecule having the sequence of Prostate carcinoma Tumor Antigen Gene-1. This invention also provides an isolated mammalian nucleic acid molecules having the sequence of Prostate Tumor Inducing Gene-1. This invention provides an isolated mammalian nucleic acid molecules having the sequence of Prostate Tumor Inducing Gene-2. Finally, this invention provides an isolated mammalian nucleic acid molecules having the sequence of Prostate Tumor Inducing Gene-3.